Obtaining the Highest Quality RNA from Blood Samples

Because of its accessibility and regenerative properties, blood is one of the most commonly used sample types for RNA isolation, especially in clinical settings. Although blood is easier to process than solid tissues because it does not require mechanical disruption, it poses unique challenges for the isolation of high quality RNA. Here we discuss these challenges and the methods available for isolating high quality RNA from blood.

Because it can be collected using relatively noninvasive procedures and is rapidly regenerated, blood is one of the only human sample types that is routinely available for RNA expression studies and biomarker discovery. As a result, blood is the most commonly used sample type in clinical research, yet it presents several challenges for RNA isolation:

1. Blood is often collected at clinical sites but processed for RNA isolation elsewhere. This leads to the requirement for RNA stabilization prior to shipping and RNA purification.
2. Blood has a complex cellular composition. The complex cellular nature of blood makes it difficult to detect differential gene expression that occurs in only a subset of cell types. The RNA-containing cells-of-interest, leukocytes, comprise <1% of the cell mass. More than 99% of the cellular blood fraction is composed of red blood cells, including immature reticulocytes, which contain high levels of globin mRNA. Globin mRNA can compromise the detection of other specific mRNAs from leukocytes.
3. Blood contains biochemicals that can compromise downstream applications. Enzyme-inhibiting compounds, such as RNase, anticoagulants, and heme, are present at relatively high concentrations in blood compared to many other tissues. If they are not adequately removed during RNA isolation, they can significantly impair downstream analyses such as microarray analysis and RT-PCR.

To enable the molecular analysis of RNA from whole blood, a stringent RNA purification method that provides high quality RNA depleted of inhibiting agents is needed. Applied Biosystems offers solutions for each step of the Blood RNA Sample Preparation Workflow (Figure 1), including sample stabilization, RNA isolation, and optional globin mRNA reduction. With a diverse array of options, researchers can choose the approach that best meets their research needs.

Blood plasma is extremely high in ribonuclease (RNase) activity, and minimizing this activity is critical to any blood RNA isolation procedure. Applied Biosystems offers two options for stabilizing the RNA in blood samples; blood samples can be drawn directly into Tempus™ Blood RNA Tubes, which contain Stabilizing Reagent, or RNA later Tissue Collection:Stabilization Solution can be added to blood samples immediately after collection.

Tempus Blood RNA Tubes
When blood is drawn into Tempus Blood RNA Tubes and mixed, the Stabilizing Reagent lyses cells almost immediately. Concurrently, cellular RNases are inactivated and RNA is selectively precipitated, leaving genomic DNA and proteins in solution. For RNA isolation, the Tempus System uses a solid-phase, silica-based purification strategy that is available in centrifugation- and vacuum-based configurations (see below, Tempus Blood RNA Isolation System). The RNA in blood collected into Tempus Blood RNA Tubes is stable for up to 5 days at room temperature, and at least 7 days at 4°C.

RNAlater Tissue Collection:Stabilization Solution
Alternatively, RNAlater Solution protects RNA from degradation without lysing blood cells. Samples in RNAlater Solution can be safely stored for up to 1 week at 25°C, or up to 1 month at 4°C. RNAlater Solution is included in several of Applied Biosystems’ blood-specific RNA purification products, including the LeukoLOCK™ Total RNA Isolation System and the RiboPure™-Blood Kit.

Both Tempus Blood RNA Tubes and RNA later Solution effectively stabilize mRNA expression profiles, and eliminate the need to isolate the RNA immediately after sample collection.

The major factors that should be considered when choosing a system for blood RNA sample preparation are blood collection volume and primary downstream application (Figures 2 and 3). Applied Biosystems offers a choice of 4 different human blood RNA isolation systems. RNA isolated with any of these systems is suitable for RT-PCR analysis.

Figure 2. Major Determinants in Choosing a Sample Preparation Method for Blood RNA.
* See Ambion GLOBINclear™-Human Whole Blood Globin Reduction Kit for the protocol
** See www.ambion.com/techlib/misc/leuko_iso.pdf for protocol
*** See www.ambion.com/techlib/misc/ribo_miRNA.pdf for protocol

Tempus Blood RNA Isolation System: Facilitates Collection and Processing of Blood for Gene Expression Analysis
The Tempus Blood RNA Tubes are designed for the collection and stabilization of whole blood for RNA isolation when real-time RT-PCR, microarrays, or other experiments that require high quality RNA will be performed. Using either spin filter (Tempus Spin RNA Isolation Kit) or vacuum filtration (Tempus 12-Port RNA Isolation Kit) methods, blood lysate is filtered through a silica-based membrane that captures the RNA. Genomic DNA, protein, and other cell debris pass through the membrane with very low retention, resulting in extremely pure RNA.

RiboPure-Blood Kit: Tailored for the Most Stringent Applications
The RiboPure-Blood Kit purifies RNA directly from whole blood without requiring fractionation of white blood cells. Separate kits are available for human or mouse/rat whole blood. By combining phenol/chloroform extraction, glass fiber filter-based purification, and DNase treatment, the RiboPure-Blood procedure removes protein, heme, genomic DNA, and RNases, resulting in high yields of exceptionally pure RNA ideal for any downstream application. The procedure is simple and fast—go from blood to RNA in <30 min. RNAlater Solution is included in the RiboPure-Blood Kit for the stabilization of RNA within the whole blood sample prior to processing for RNA. The kit is ideal for small sample volumes, but is also available in custom formats (e.g., for larger sample volumes).

LeukoLOCK Total RNA Isolation System: Specifically Captures White Blood Cells for Array Analysis
The LeukoLOCK Total RNA Isolation System is an innovative method for cellular fractionation of whole blood and total RNA stabilization and extraction from the leukocyte population. The LeukoLOCK System is optimized for use with human blood. Blood is a storehouse of cellular information; however, the presence of globin mRNA in RNA prepared from whole blood can interfere with microarray-based expression profiling applications. The LeukoLOCK System employs filter-based leukocyte-depletion technology to capture leukocytes from whole blood and RNAlater Solution to stabilize the cells on the filter. By excluding red blood cells from the RNA purification, the RNA is inherently depleted of globin mRNA, resulting in improved microarray results.

MagMAX™-96 Blood RNA Isolation Kit: A High Throughput Solution for Viral and Total RNA
The MagMAX-96 Blood RNA Isolation Kit is designed for rapid (<1 hour), isolation of RNA, including viral RNA, from mammalian whole blood in 96-well plate format. The cells are lysed, and RNA is purified from the lysate using Ambion MagMAX magnetic bead-based technology. Magnetic beads bind RNA more efficiently than glass fiber filter methods, resulting in higher RNA yields. Furthermore, the RNA yields are more consistent from experiment to experiment, ensuring more reliable results from your downstream analyses. Viral and total RNA obtained using the MagMAX-96 Blood Kit can be used directly in real-time RT-PCR for viral RNA identification. However, since the kit does not use stabilized blood, it is not suited for gene expression analysis. RNA purification using the MagMAX-96 Blood Kit can easily be automated with the MagMAX Express Particle Processors.

Up to 70% of the mRNA (by mass) in whole blood consists of globin transcripts from red blood cells (RBCs). These “unwanted” globin transcripts effectively dilute the mRNA population and decrease the sensitivity of detecting less abundant mRNAs using microarray technology. In array analyses, the high percentage of globin mRNA from blood samples has been shown to:

• Decrease present calls
• Decrease call concordance
• Increase signal variation

To address this problem, Applied Biosystems offers two different approaches that are both compatible with RNA amplification and labeling protocols (e.g., Ambion MessageAmp™ Kits):

LeukoLOCK Total RNA Isolation System
The LeukoLOCK System preserves gene expression profiles, while eliminating >90% of globin mRNA through the exclusion of RBCs from the sample (see above, LeukoLOCK Total RNA Isolation System).

GLOBINclear™-Human Whole Blood Globin Reduction Kit
The GLOBINclear Kit can be used to remove >95% of unwanted globin mRNA from whole blood total RNA. Instead of using harsh RNase H treatments that can degrade mRNA and alter expression profiles, this kit employs a novel hybridization technology that takes advantage of the strength of biotin/streptavidin binding, the specificity of nucleic acid hybridization, and the convenience of magnetic separations. These features make the kit an ideal solution for removing globin mRNA from blood RNA that has already been isolated with the RiboPure-Blood Kit or the Tempus Blood RNA System.

Many current RNA purification procedures rely on organic extraction followed by alcohol precipitation or adsorption of nucleic acid molecules on a glass-fiber filter or silica matrix. Unfortunately, the former procedure requires alcohol precipitation, which is time consuming and inefficiently recovers small RNAs, and the latter procedure typically results in the loss of substantial amounts of small RNA (<200 bases), including microRNAs (miRNAs) and siRNAs.

Applied Biosystems offers several kits that use optimized buffers and washes to isolate small RNAs. For blood miRNA isolation, we have developed supplemental protocols for the RiboPure-Blood Kit and LeukoLOCK System to adapt them for miRNA analysis.